Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana

In order to obtain recombinant antibody fragments that bind the cell-cycle protein CDC2a from Arabidopsis thaliana (CDC2aAt), two phage display librar ies of single-chain variable (scFv) fragments were constructed. One library was derived from mice immunized with recombinant CDC2aAt N-terminally fuse d to a His(6)-tag (His-CDC2aAt) and the other was made out of an anti-PSTAI RE hybridoma cell line. Six specific His-CDC2aAt-binding phage clones (3D1, 3D2, 3D10, 3D25, 4D21 and 4D47) were isolated by panning. The isolated mon oclonal phage clones, as well as the soluble scFv fragments produced in the periplasm of Escherichia coli, bind His-CDC2aAt in ELISA and on Western bl ots. Moreover, four clones (3D1, 3D2, 3D10 and 4D21) detect specifically CD C2aAt from Arabidopsis cell suspensions on Western blots. Clone 4D21 binds the PSTAIRE epitope, whereas the 3D1, 3D2 and 3D10 clones bind, as yet unid entified, epitopes of CDC2aAt. Furthermore, the accumulation and antigen-bi nding activity of these scFv fragments in a reducing environment were asses sed. No interaction could be shown between the scFv fragments and CDC2aAt i n a yeast two-hybrid assay. However, after transient expression of the scFv fragments in the cytosol of tobacco leaves, three of six scFv fragments (3 D1, 3D2 and 3D10) accumulated in the plant cytosol and ELISA results indica te that these scFv fragments retained antigen-binding activity.